Exam 4 Chapter 16 Notecards

Microbiology 310 with Reynolds at University of Tennessee - Knoxville

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By: April Besing
Textbook:

Microbiology: An Evolving Science (Second Edition)
Created: 2010-05-03
Size: 76 flashcards
Views: 71

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Chapter 16

Biotechnology and Industrial Microbiology

Restriction enzymes

are enzymes which cleave viral DNA at specific points to protect the cell from virus infection; they are used in vitro to carry out genetic engineering.

Reverse transcriptase

, an RNA-dependent DNA polymerase that uses a viral RNA genome as a template to synthesize a DNA copy, uses processed mRNA as a template for cDNA.

1 Key Developments in Recombinant DNA Technology

Restriction enzymes are enzymes which cleave viral DNA at specific points to protect the cell from virus infection;...
From these segments of DNA, recombinant plasmid were formed relatively quickly.
However, cloning eukaryotic DNA was not as easy: pre-mRNA must be processed and prokaryotes lack the machinery to do...
Southern blotting technique enables the detection of specific DNA fragments from a mixture of DNA molecules.

Southern blotting technique

enables the detection of specific DNA fragments from a mixture of DNA molecules.

3 Step cycle

denaturation, annealing, elongation

Restriction enzymes are enzymes which cleave viral DNA at specific points to protect the cell from virus infection; they are used in vitro to carry out genetic engineering.

They recognize DNA at specific sites known as recognition sites.
Type I and III cleave DNA at a set distance from the recognition site, while type II cleave at that site.
When it cleaves the DNA some endonucleases leave sticky ends , or a single strand sequence on either end of the double stranded segment.

2 Polymerase Chain Reaction

3 Step cycle: denaturation, annealing, elongation.
Uses DNA oligonucleotides as primers, which are annealed.
Taq polymerase, a heat tolerant polymerase, is used to elongate the DNA.
2 main uses
The procedure has numerous uses in several fields: isolation of certain genes, diagnostic tests, and DNA fingerprints.

However, cloning eukaryotic DNA was not as easy: pre-mRNA must be processed and prokaryotes lack the machinery to do this process.

Reverse transcriptase , an RNA-dependent DNA polymerase that uses a viral RNA genome as a template to synthesize a DNA copy, uses processed mRNA as a template for cDNA.

Real-time PCR

is quantitative?how much of a template is present in a sample.

Southern blotting technique enables the detection of specific DNA fragments from a mixture of DNA molecules.

First, segments are separated by size by gel electrophoresis.
Then, the DNA is denatured and moved to a filter.
The filter is then bathed with a radioactive probe.
When exposed, the radioactive probe leaves a silver band, indicating the DNA.

Gel electrophoresis

is a technique that sorts molecules according to charge and size.

Cloning vector,

a DNA molecule that can replicate independent of the host chromosome and transport a piece of inserted foreign DNA, such as a gene, into a recipient cell.

3 Gel Electrophoresis

Gel electrophoresis is a technique that sorts molecules according to charge and size.
When a DNA fragment is placed near a negative pole, it will migrate toward the positive pole at a rate which is directly related to its size.
DNA is usually cut with restriction enzymes before the process.

2 main uses

Isolation of a particular DNA sequence, not quantitative.
Real-time PCR is quantitative?how much of a template is present in a sample. Accomplished by adding a fluorescently labeled probe to the reaction mixture and measuring the signal through the first few cycles.

Origin of replication

allows the plasmid to replicate independently of the chromosome.

Multicloning site

or Polylinker.

Cosmid

is a plasmid vector with a lambda phage cos sites that can be packaged in a phage capsid; it is useful for cloning large DNA fragments.

4 Cloning Vectors and Creating Recombinant DNA

Recombinant DNA technology depends on the propagation of many copies of the nucleotide sequence of choice.
Cloning vector, a DNA molecule that can replicate independent of the host chromosome and transport a piece of inserted foreign DNA, such as a gene, into a...
All vectors share three common features: origin of replication, a region of DNA that bears unique restriction sites (multicloning site or polylinker), and a...

Genomic Library

is a collection of clones that contains fragments that represent the complete genome of an organism.

5 Construction of Genomic Libraries

Genomic Library is a collection of clones that contains fragments that represent the complete genome of an organism.
To select a desired gene from a library, something about its function of the target gene or genetic element must be known.
Phenotypic rescue is the identification of a cloned gene based on its ability to complement a genetic deficiency in...
If a genomic library is prepared from a eukaryotic cell, a cDNA library is usually constructed to avoid intons.

Phenotypic rescue

is the identification of a cloned gene based on its ability to complement a genetic deficiency in the host cell.

All vectors share three common features: origin of replication, a region of DNA that bears unique restriction sites (multicloning site or polylinker), and a selectable marker.

Plasmids
Phage Vector
Cosmids
Artificial Chromosomes

Plasmids

Replicate autonomously and are easily purified.
Introduced to microbes via conjugation or transformation.
Origin of replication allows the plasmid to replicate independently of the chromosome.
Selectable marker codes for a protein which is needed for the cell to survive under...
Multicloning site or Polylinker. Is a region of DNA on a cloning vector that has a number...

Phage Vector

Once foreign DNA is inserted into the phage genome, it is packaged into viral capsids and used to infect other cells and then the lytic cycle further propagates the genome.

Origin of replication allows the plasmid to replicate independently of the chromosome.

Some plasmids have two origins of replication, recognizable by different hosts. These are known as shuttle vectors , because they can move from one host to another.

Cosmids

Cosmid is a plasmid vector with a lambda phage cos sites that can be packaged in a phage capsid; it is useful for cloning large DNA fragments.
Do not exist in nature, but contain features from phages and plasmids.

Artificial Chromosomes

Are especially useful when cloning large fragments of DNA.
Bacterial artificial chromosomes
Yeast artificial chromosomes
BACs are more stable than YACs

Do not exist in nature, but contain features from phages and plasmids.

MCS from plasmids
Cos site from phage, allowing it to become circular.

6 Introducing Recombinant DNA into Host Cells

Host microbes that have been engineered to lack restriction enzymes and a recombination enzyme usually make better hosts because it is more likely that the host DNA will be preserved.
Transformation and electroportation are the most common techniques of inserting DNA.

Classical method

recombinant DNA technology

7 Expressing Foreign Genes in Host Cells

When a gene from one organism is cloned into another, it is said to be a heterologous gene.
These problems have been largely overcome by the use of expression vectors , which contain the proper transcription and translation start signals and the convenient multicloning sites.
Example: Somatostatin gene.

Site-directed mutagenesis

, where the nucleotide sequence of a specific gene can be altered.

Transformation and electroportation are the most common techniques of inserting DNA.

Electroportation is a technique that is simple and has wide applicability.

Cells are mixed with recombinant DNA and exposed to high voltage electricity, which makes the plasma membrane temporarily permeable.

When a gene from one organism is cloned into another, it is said to be a heterologous gene.

To be transcribed the heterologous gene must have a recognizable promoter.
Translation must have leader sequences and mRNA modifications for proper ribosome binding.

8 Microorganisms used in Industrial Microbiology

Utilizes the capacity of microbes to synthesize compounds with wide applicability, known as natural products.
First step is to find a suitable microorganism, which is genetically stable, easy to maintain and grow, and well suited for extraction of the desired products.

These problems have been largely overcome by the use of expression vectors , which contain the proper transcription and translation start signals and the convenient multicloning sites.

Some contain regulatory regions of the lac operon, which allows for control of expression in the same manner as the operon.

forced evolution

and adaptive mutation which use the application of specific environmental stresses which force microorganisms to mutate and adapt.

In vitro evolution

starts with purified nucleic acids rather than a whole organism.

High-throughput screening

enables the rapid selection of a single desirable molecule from tens of thousands of compounds.

9 Microorganism growth in Controlled Environments

Industrial fermentations require the development of appropriate culture media and the...
Microorganisms are often grown in stirred fermenters ranging from 3-100,000 liters.
Frequently, a critical component is constantly added? continuous feed ?so that the microbes...
Continuous growth is not always favorable, since in some cases the product of interest is not...
Microbial products are often classified as primary and secondary metabolites.

First step is to find a suitable microorganism, which is genetically stable, easy to maintain and grow, and well suited for extraction of the desired products.

Finding Microorganisms in Nature? bioprospecting , or the hunting of new microorganisms.
Genetic Manipulation of Microorganisms

Primary metabolites

consist of compounds related to the synthesis of microbial cells during balanced growth.

Finding Microorganisms in Nature? bioprospecting , or the hunting of new microorganisms.

Less than 1% of the microbial species estimated to exist have been isolated or cultured.

Genetic Manipulation of Microorganisms

Classical method: recombinant DNA technology
Mutagenesis
Protoplast fusion
Transfer of genetic Information Between Different Organisms

Mutagenesis

Chemical, ultraviolet light, and transposon mutagenesis.
Site-directed mutagenesis , where the nucleotide sequence of a specific gene can be altered.

Protoplast fusion

Protoplasts, or cells without walls, are combined with the other cells.

Transfer of genetic Information Between Different Organisms

Part of combinatorial biology .
When functional genes from one organism are transcribed and translated in another, it is known as heterologous gene expression.
Modification of Gene Expression
Protein Evolution

When functional genes from one organism are transcribed and translated in another, it is known as heterologous gene expression.

Enables the production of certain proteins and peptides without the contamination by other products which would be produced in the normal organism.
Only biologically active stereoisomers are produced.

Modification of Gene Expression

Modify gene regulators to result in overproduction
Alteration of metabolic processes? metabolic pathway engineering

Protein Evolution

Involves forced evolution and adaptive mutation which use the application of specific environmental stresses which force microorganisms to mutate and adapt.
In vitro evolution starts with purified nucleic acids rather than a whole organism. DNA templates are transcribed in vitro by phage RNA polymerase. Then reverse transcriptase comes in. After propagating the cDNA, the proteins are screened.

In vitro evolution starts with purified nucleic acids rather than a whole organism. DNA templates are transcribed in vitro by phage RNA polymerase. Then reverse transcriptase comes in. After propagating the cDNA, the proteins are screened.

High-throughput screening enables the rapid selection of a single desirable molecule from tens of thousands of compounds.

Employs robotics and computer analysis.

Secondary metabolites

usually accumulate during the phase of nutrient limitation or waste product accumulation.

10 Major Products of Industrial Microbiology

Antibiotics
Amino Acids
Organic Acids
Biopolymers
Biosurfactants
Biofuels
Bioconservation Processes

Microorganisms are often grown in stirred fermenters ranging from 3-100,000 liters.

Medium must be sterile, and pH and aeration must be monitored.

Microbial products are often classified as primary and secondary metabolites.

Primary metabolites consist of compounds related to the synthesis of microbial cells during balanced growth.
Secondary metabolites usually accumulate during the phase of nutrient limitation or waste product accumulation.

Primary metabolites consist of compounds related to the synthesis of microbial cells during balanced growth.

Amino acids, nucleotides, fermentation end products.

Secondary metabolites usually accumulate during the phase of nutrient limitation or waste product accumulation.

Most antibiotics and mycotoxins fall into this class.

Antibiotics

Penicillin is a prime example of the effects of environmental controls on the end products of fermentation.

11 Recombinant DNA Technology in Agriculture

Allows for the genetic modification of plants for resistance to infection.
Another application is the use of bacteria, viruses, and fungi as bioinsecticides and biopesticides.

Penicillin is a prime example of the effects of environmental controls on the end products of fermentation.

If a slow feed of glucose is maintained, then there is more penicillin in the end.
If a specific penicillin is desired, then a particular precursor is added.
pH is maintained neutral for maximum yield

Amino Acids

Lysine and glutamic acid are used as nutritional supplements.
Production is usually carried out by regulatory mutants with a diminished capacity to stop synthesis of an amino acid or intermediate.

Organic Acids

Citric, acetic, lactic, fumaric, and gluconic acids
The production of citric acid depends on the limitation of trace metals to stop growth at a specific point.
High sugar concentrations are used, regulation of the glycolytic pathway and the carboxylic acid cycle is essential.
After active growth phase, accumulation increases.

Biopolymers

Dextrans?used as blood expanders and absorbents
Erwinia polysaccharides are used in paints.
Polyesters are a feedstock for specialty plastics
Cellulose microfibrils are used as food thickeners
Polysaccharides are used by the oil industry as drilling mud additives
Xanthan polymers enhance oil recovery by improving water flooding and the displacement of oil

Biosurfactants

Amphiphilic molecules which partition the interface between fluids of different polarity.
Used for emulsification, increasing detergency, wetting and phase dispersion and solubilization.
Important to bioremediation, oil spill dispersion and enhanced oil recovery.
Most widely produced are glycolypids.
Have an antibacterial and antifungal activity due to their ability to disrupt the plasma membrane.

Biofuels

The polysaccharides of interest can be fermented only by bioengineered organisms.
Also researching ways to degrade cellulose and hemicellulose.

Bioconservation Processes

Also known as microbial transformation or biotransformation are minor changes in molecules, such as insertions of hydroxyl or keto functions, or the...
The microbe acts as a biocatalyst.
More efficiently stereochemically than chemical procedures. Also, enzymes carry out specific reactions in mild conditions and larger water-insoluble...

Allows for the genetic modification of plants for resistance to infection.

Example: Ti plasmid.

12 Microbes as Products

Diatoms have aroused the interest of nanotechnology, because of their silica shells, which can be converted with magnesium
Magnetotactic bacteria, which convert iron to magnetite, are also of interest to nanotechnology, due to their perfectly formed structures.
Biosensor production is a field in which microorganisms are linked with electrodes and biological reactions are converted into electric currents.

thuringiensis is the most widely used.

Toxin is used as an insecticide known as Bt and the genes can be used to genetically modify plants.
Does not accumulate in soil or in nontarget animals, but is quickly lost to the environment by microbial or abiotic degradation.
Bt is nontoxic to mammals and is not an allergen in humans.

Example

Ti plasmid

Biosensor production is a field in which microorganisms are linked with electrodes and biological reactions are converted into electric currents.

Used to measure components in beer, monitor pollutants, detect flavor compounds in food and study environmental processes such as changes in biofilm concentration gradients.
New biosensors have been developed from a immunochemical-based detection systems
Use the streptavidin-biotin recognition system.

Biosensor

production is a field in which microorganisms are linked with electrodes and biological reactions are converted into electric currents.

New biosensors have been developed from a immunochemical-based detection systems

Used to detect pathogens, herbicides, toxins, proteins and DNA.

About this deck

By: April Besing
Textbook:

Microbiology: An Evolving Science (Second Edition)
Created: 2010-05-03
Size: 76 flashcards
Views: 71

About StudyBlue

“Simply amazing. The flash cards are smooth, there are many different types of studying tools, and there is a great search engine. I praise you on the awesomeness.”
Dennis